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Science 20 March 1998:
Vol. 279. no. 5358, pp. 1958 - 1961
DOI: 10.1126/science.279.5358.1958
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Reports

Redesigning Enzyme Topology by Directed Evolution

Gavin MacBeath, Peter Kast, Donald Hilvert *

Genetic selection was exploited in combination with structure-based design to transform an intimately entwined, dimeric chorismate mutase into a monomeric, four-helix-bundle protein with near native activity. Successful reengineering depended on choosing a thermostable starting protein, introducing point mutations that preferentially destabilize the wild-type dimer, and using directed evolution to optimize an inserted interhelical turn. Contrary to expectations based on studies of other four-helix-bundle proteins, only a small fraction of possible turn sequences (fewer than 0.05 percent) yielded well-behaved, monomeric, and highly active enzymes. Selection for catalytic function thus provides an efficient yet stringent method for rapidly assessing correctly folded polypeptides and may prove generally useful for protein design.

The Scripps Research Institute, Departments of Chemistry and Molecular Biology, 10550 North Torrey Pines Road, La Jolla, California, 92037, USA.
*   To whom correspondence should be addressed at Department of Chemistry, Swiss Federal Institute of Technology (ETH), CH-8092 Zürich, Switzerland. E-mail: hilvert{at}org.chem.ethz.ch

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